Carotenoid is a natural pigment widely existent in the natural world, and is a polyene pigment having a color in the range of yellow to red or purple. Astaxanthin is one type of naturally-occurring carotenoid and exists in a free state or as an ester, or exists as various types of pigment proteins as a result of being bonded therewith.
Astaxanthin is widely used as a coloring agent for fishes and chicken's eggs. Astaxanthin is also permitted to be used as a food additive and is widely used in fat and oil processed foods, protein foods, aqueous liquid foods and the like. Astaxanthin further has an anti-oxidation activity against peroxidation of lipid excited by a free radical, a singlet oxygen erasing action which can be several hundred times stronger than that of α-tocopherol or the like, and therefore is expected to be used in functional foods, cosmetics, and pharmaceutical drugs by utilizing the strong anti-oxidation activity thereof.
Astaxanthin is distributed widely in the natural world in, for example, fishes such as salmon, trout and red sea bream, etc.; and crustaceans such as crab, shrimp, krill, etc. Astaxanthin is also produced by bacteria belonging to Agrobacterium, Brevibacterium and Paracoccus; and microorganisms including Haematococcus pluvialis, Phaffia yeast and the like. Carotenoid such as astaxanthin, zeaxanthin or the like is industrially produced by a chemical synthesis method, but naturally-occurring carotenoid is desired from the aspect of safety.
With such a background, many methods for producing carotenoids containing astaxanthin especially derived from algae or microorganisms which are considered to be suitable to mass production have been reported.
For example, the following method for producing carotenoid from a Haematococcus alga has been reported (Japanese Laid-Open Patent Publication No. H 11-56346). A cystocyte of a post-culture alga is treated with heated acetone to elute chlorophyll, which is a contaminant. Then, the cystocyte is spray-dried, and carotenoid is extracted from the resultant dry cells with ethanol. However, a composition obtained by such a method still contains many contaminants derived from organisms, and is not satisfactory in terms of 1) the carotenoid content, 2) the astaxanthin content, and the like.
In order to obtain a composition containing astaxanthin at a high content, the following method, for example, has been reported (Japanese Laid-Open Patent Publication No. 2002-218994). A crude xanthophylls obtained in conformity with the above-described method is acted on by lipase in the presence of water to decompose a neutral lipid, which is one contaminant. The lipase enzyme-treated liquid is decomposed into oil and water. From the separated oil layer, free fatty acid is separated from astaxanthin by distillation, and the astaxanthin is concentrated and purified. However, even after such complicated treating steps, a composition containing astaxanthin at a ratio of 30% or higher has not been obtained.
A method of obtaining astaxanthin contained at a ratio of 0.5 to 60% using a supercritical fluid extraction method (Japanese Laid-Open Patent Publication No. 2004-41147) has been reported. However, it is inevitable that an astaxanthin fraction of a content less than the targeted content is produced as a sub-product. In order to discard, or increase the astaxanthin content of, such a fraction, another concentration operation is required. Therefore, this production method is not satisfactory, in terms of the simplicity and cost, as an industrial method for producing highly pure carotenoid containing a high content of astaxanthin with little contaminants derived from organisms.
As a method using Phaffia yeast, the following method has been reported (Japanese Laid-Open Patent Publication No. H 10-276721). A crushed bacterial cell of the yeast is treated with extraction using an organic solvent, and the oil-like crude extraction obtained by concentrating the extract solution is purified by ion exchange chromatography, adsorption chromatography or the like to obtain astaxanthin. However, this method is performed by a non-industrial technique of purifying the crude solution of low concentration astaxanthin through a plurality types of column chromatography.
As another method, the following method has also been reported (Japanese Laid-Open Patent Publication No. 2004-208504). A bacterial cell after the culturing of Phaffia yeast is treated with extraction using acetone, and the resultant extract is concentrated to obtain a crude oil-like substance. A hydrocarbon-based solvent is added to this crude oil-like substance to deposit crystals. This method is highly simple, but the obtained composition contains carotenoid at a content of merely about 70 to 73% (the content of astaxanthin is merely 36 to 42%). Due to such a low content, this method is not satisfactory as a method for producing highly pure carotenoid with little contaminants derived from organisms. This method is not satisfactory also for the reason that there is a concern that acetone and the hydrocarbon-based solvent may remain in carotenoid.
As methods using E-396 strain (FERM BP-4283: deposited on Apr. 27, 1993 (date of original deposition), International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, Higashi 1-1-1, Tsukuba-shi, Ibaraki-ken, Japan)), which is a bacterial strain for producing astaxanthin, adonixanthin and the like, the following methods have been reported: a method of extracting by contacting a cyclic hydrophilic organic compound, which invokes a safety concern regarding the use in food production, to the bacterial cell (Japanese Laid-Open Patent Publication No. H 7-242621); and a method of using a supercritical fluid extraction method like document 3 (Japanese Laid-Open Patent Publication No. H 8-89280). A method of contacting E-396 strain to a water-soluble organic solvent, a non-polar solvent and water to perform liquid-liquid extraction (Japanese Laid-Open Patent Publication No. H 8-253695) has also been reported.
Under these circumstances, a method for industrially producing highly pure carotenoid containing astaxanthin at a high content, which is simple without requiring any special facilities or complicated operations and which uses a solvent safe for food production, is strongly desired to be established.